Timely and accurate regulation of gene expression is required for proper growth, development, and response to environmental stimuli. A complete understanding of the mechanisms employed to regulate gene expression is necessary to combat the aberrant regulation that underlies many human developmental conditions and ailments, including cancer. The long-term goals of this proposal are to determine the mechanics of RNA synthesis by DNA-dependent multi-subunit RNA polymerases (RNAPs), the regulation imposed on RNAP by conserved protein factors and select template sequences, and to specifically characterize the available mechanisms to halt RNA synthesis and terminate transcription. The proposed experiments take advantage of both an in vivo and in vitro established archaeal transcription system. Archaea offer the advantages of far less complexity but homology in many features of human molecular biology, specifically conservation of RNAP structure and function. We will select, generate, purify and characterize variant RNAPs with modified transcription termination phenotypes. We will further characterize factor-dependent termination, polarity, and the role of such regulation in Archaea. The results so obtained will determine the structures and sequences that support transcription elongation complex stability, describe the mechanics of termination and allow comparisons of termination mechanisms in each Domain, and open a new field of study, archaeal factor-dependent termination. PUBLIC HEALTH RELEVANCE: A complete understanding of the mechanisms employed to regulate gene expression is necessary to combat the aberrant regulation that underlies many human developmental conditions and ailments, including cancer. The long-term goals of this proposal are to determine the mechanics of RNA synthesis by DNA-dependent multi-subunit RNA polymerases (RNAPs), the regulation imposed on RNAP by conserved protein factors and select template sequences, and to specifically characterize the available mechanisms to halt RNA synthesis and terminate transcription. The results so obtained will determine the structures and sequences that support transcription elongation complex stability, describe the mechanisms of termination and allow comparisons of termination mechanisms in each Domain, and open a new field of study, archaeal factor-dependent termination.